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1.
Hum Mol Genet ; 33(5): 448-464, 2024 Feb 18.
Article in English | MEDLINE | ID: mdl-37975905

ABSTRACT

Biallelic mutations in interphotoreceptor matrix proteoglycan 2 (IMPG2) in humans cause retinitis pigmentosa (RP) with early macular involvement, albeit the disease progression varies widely due to genetic heterogeneity and IMPG2 mutation type. There are currently no treatments for IMPG2-RP. To aid preclinical studies toward eventual treatments, there is a need to better understand the progression of disease pathology in appropriate animal models. Toward this goal, we developed mouse models with patient mimicking homozygous frameshift (T807Ter) or missense (Y250C) Impg2 mutations, as well as mice with a homozygous frameshift mutation (Q244Ter) designed to completely prevent IMPG2 protein expression, and characterized the trajectory of their retinal pathologies across postnatal development until late adulthood. We found that the Impg2T807Ter/T807Ter and Impg2Q244Ter/Q244Ter mice exhibited early onset gliosis, impaired photoreceptor outer segment maintenance, appearance of subretinal deposits near the optic disc, disruption of the outer retina, and neurosensorial detachment, whereas the Impg2Y250C/Y250C mice exhibited minimal retinal pathology. These results demonstrate the importance of mutation type in disease progression in IMPG2-RP and provide a toolkit and preclinical data for advancing therapeutic approaches.


Subject(s)
Proteoglycans , Retinitis Pigmentosa , Humans , Animals , Mice , Adult , Proteoglycans/genetics , Retina , Mutation , Retinitis Pigmentosa/genetics , Disease Progression
2.
Proc Natl Acad Sci U S A ; 120(2): e2213418120, 2023 01 10.
Article in English | MEDLINE | ID: mdl-36598946

ABSTRACT

Human pluripotent stem cell (hPSC)-derived retinal organoids (ROs) can efficiently and reproducibly generate retinal neurons that have potential for use in cell replacement strategies [Capowski et al., Development 146, dev171686 (2019)]. The ability of these lab-grown retinal neurons to form new synaptic connections after dissociation from ROs is key to building confidence in their capacity to restore visual function. However, direct evidence of reestablishment of retinal neuron connectivity via synaptic tracing has not been reported to date. The present study employs an in vitro, rabies virus-based, monosynaptic retrograde tracing assay [Wickersham et al., Neuron 53, 639-647 (2007); Sun et al., Mol. Neurodegener. 14, 8 (2019)] to identify de novo synaptic connections among early retinal cell types following RO dissociation. A reproducible, high-throughput approach for labeling and quantifying traced retinal cell types was developed. Photoreceptors and retinal ganglion cells-the primary neurons of interest for retinal cell replacement-were the two major contributing populations among the traced presynaptic cells. This system provides a platform for assessing synaptic connections in cultured retinal neurons and sets the stage for future cell replacement studies aimed at characterizing or enhancing synaptogenesis. Used in this manner, in vitro synaptic tracing is envisioned to complement traditional preclinical animal model testing, which is limited by evolutionary incompatibilities in synaptic machinery inherent to human xenografts.


Subject(s)
Pluripotent Stem Cells , Retina , Animals , Humans , Reactive Oxygen Species , Retina/physiology , Retinal Ganglion Cells , Organoids , Cell Differentiation
3.
Stem Cell Reports ; 17(11): 2409-2420, 2022 11 08.
Article in English | MEDLINE | ID: mdl-36206764

ABSTRACT

Interphotoreceptor matrix proteoglycan 2 (IMPG2) mutations cause a severe form of early-onset retinitis pigmentosa (RP) with macular involvement. IMPG2 is expressed by photoreceptors and incorporated into the matrix that surrounds the inner and outer segments (OS) of rods and cones, but the mechanism of IMPG2-RP remains unclear. Loss of Impg2 function in mice produces a mild, late-onset photoreceptor phenotype without the characteristic OS loss that occurs in human patients. We generated retinal organoids (ROs) from patient-derived induced pluripotent stem (iPS) cells and gene-edited embryonic stem cells to model human IMPG2-RP in vitro. All ROs harboring IMPG2 mutations lacked an OS layer, in contrast to isogenic controls. Subsequent protein analyses revealed that this phenotype arises due to a loss of IMPG2 expression or its inability to undergo normal post-translational modifications. We hypothesized that loss of IMPG2 function destabilizes the interphotoreceptor matrix and renders the OS vulnerable to physical stressors, which is accentuated in the tissue culture environment. In support of this mechanism, transplantation of IMPG2 mutant ROs into the protected subretinal space of immunocompromised rodents restored OS production. Beyond providing a robust platform to study IMPG2-RP, this human RO model system may serve a broader role in honing strategies to treat advanced photoreceptor-based diseases.


Subject(s)
Organoids , Retinitis Pigmentosa , Humans , Mice , Animals , Organoids/metabolism , Reactive Oxygen Species/metabolism , Eye Proteins/genetics , Proteoglycans/genetics , Retinitis Pigmentosa/genetics , Retina/metabolism , Mutation , Retinal Cone Photoreceptor Cells/metabolism , Phenotype
4.
Nat Commun ; 12(1): 6324, 2021 11 03.
Article in English | MEDLINE | ID: mdl-34732726

ABSTRACT

Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3' UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.


Subject(s)
Cardiomyopathies/genetics , Cardiomyopathies/metabolism , Gain of Function Mutation , Mutation , RNA Splicing , RNA-Binding Proteins/genetics , Ribonucleoproteins/metabolism , Cardiomyopathy, Dilated/genetics , DEAD-box RNA Helicases , DNA Helicases , Gene Knockdown Techniques , Humans , Induced Pluripotent Stem Cells/metabolism , Male , Mutation, Missense , Poly-ADP-Ribose Binding Proteins/metabolism , Proto-Oncogene Proteins , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism
5.
Development ; 146(1)2019 01 09.
Article in English | MEDLINE | ID: mdl-30567931

ABSTRACT

Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. Although nearly all such methods yield at least partial segments of retinal structure with a mature appearance, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to understand better the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation.


Subject(s)
Organoids/cytology , Pluripotent Stem Cells/cytology , Retina/cytology , Cell Differentiation , Cell Line , Cell Proliferation , Cell Shape , Ependymoglial Cells/cytology , Ependymoglial Cells/metabolism , Humans , Interneurons/cytology , Interneurons/metabolism , Models, Biological , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/ultrastructure , Reproducibility of Results , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/metabolism , Synapses/metabolism , Tomography, Optical Coherence
6.
Methods Mol Biol ; 1768: 349-362, 2018.
Article in English | MEDLINE | ID: mdl-29717453

ABSTRACT

Genome editing holds great promise for experimental biology and potential clinical use. To successfully utilize genome editing, it is critical to sensitively detect and quantify its outcomes: homology-directed repair (HDR) and nonhomologous end joining (NHEJ). This has been difficult at endogenous gene loci and instead is frequently done using artificial reporter systems. Here, we describe a droplet digital PCR (ddPCR)-based method to simultaneously measure HDR and NHEJ at endogenous gene loci. This highly sensitive and quantitative method may significantly contribute to a better understanding of DNA repair mechanisms underlying genome editing and to the improvement of genome editing technology by allowing for efficient and systematic testing of many genome editing conditions in parallel.


Subject(s)
DNA End-Joining Repair/genetics , DNA/isolation & purification , Genetic Loci/genetics , Polymerase Chain Reaction/methods , Recombinational DNA Repair/genetics , DNA/genetics , Gene Editing/methods , HEK293 Cells , Humans , Induced Pluripotent Stem Cells , Polymerase Chain Reaction/instrumentation
7.
Nat Commun ; 7: 12009, 2016 07 01.
Article in English | MEDLINE | ID: mdl-27363581

ABSTRACT

Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants.


Subject(s)
Bacterial Proteins/metabolism , CRISPR-Cas Systems , Endonucleases/metabolism , Gene Editing/methods , Protein Domains , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Estrogen/metabolism , Receptors, Glucocorticoid/metabolism , CRISPR-Associated Protein 9 , Clustered Regularly Interspaced Short Palindromic Repeats , Epigenesis, Genetic , Estrogen Antagonists/pharmacology , Gene Expression Regulation , Glucocorticoids/pharmacology , Plasmids , Protein Binding , Receptors, Cytoplasmic and Nuclear/drug effects , Receptors, Estrogen/drug effects , Receptors, Glucocorticoid/drug effects , Staphylococcus aureus , Streptococcus pyogenes , Tamoxifen/analogs & derivatives , Tamoxifen/pharmacology
8.
Sci Rep ; 6: 23549, 2016 Mar 31.
Article in English | MEDLINE | ID: mdl-27030102

ABSTRACT

Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR-based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator-like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.


Subject(s)
DNA Breaks, Double-Stranded , DNA Breaks, Single-Stranded , DNA End-Joining Repair , Gene Editing , Genome, Human , Recombinational DNA Repair , Transcription Activator-Like Effector Nucleases/genetics , Biological Assay , CRISPR-Cas Systems , Cell Line , Genetic Loci , HEK293 Cells , HeLa Cells , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Plasmids/chemistry , Plasmids/metabolism , Polymerase Chain Reaction , Transcription Activator-Like Effector Nucleases/metabolism , Transfection
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